Integrins, extracellular matrix molecules, and cytoskeletal proteins contribute to cell adhesion and migration, and they participate in cell surface control of tissue organization, gene expression, and growth. [unreadable] We are addressing the following general questions:[unreadable] 1. How are integrins, the extracellular matrix, and the cytoskeleton integrated and coordinated to produce cell migration?[unreadable] 2. What are the subcellular structures and signaling pathways involved in biological functions such as migration?[unreadable] [unreadable] We are using a variety of cell and molecular biology approaches to address these questions, including biochemical analyses, fluorescent chimeras, fluorescence time-lapse microscopy, and mutational analysis. We have generated a variety of fluorescent molecular chimeras and mutants of cytoskeletal proteins, including paxillin, vinculin, talin, and tensin, as part of an ongoing program to analyze their functions in integrin-mediated processes. We have been focusing particularly on functions of integrins and associated molecules in the mechanisms of cell migration. [unreadable] [unreadable] Nonmuscle cellular myosins and actin are thought to play crucial roles in cell migration and in many developmental and wound repair processes, but the roles of the major myosin IIA gene were not clear. Our laboratory has undertaken cell biological characterization of cells lacking this predominant myosin of nonmuscle cells. Our findings relating to cell contractility were not surprising: knockout or siRNA knockdown cells and cells treated with the myosin II inhibitor blebbistatin show losses of focal adhesions and actin stress fibers, and they become defective in their ability to contract fibrin gels. Unexpectedly, we found that myosin IIA-deficient cells show enhanced random cell migration, with hyperactive cell protrusiveness and elaborate leading lamellae. The regulatory GTPase Rac-1 and its activator Tiam-1 become localized at the leading edge of these migrating cells, and they display elevated Rac activation. We explored the mechanism and found that microtubules provide a link between myosin and Rac activation, and disruption of microtubules reverses the hyper-migratory phenotype and restores normal morphology. The microtubules of cells missing myosin IIA show enhanced stability and become abundant near the edges of lamellae; we speculate that they may transport Tiam-1 to the leading edge. Moreover, transfection of myosin IIA into a cell line that normally lacks myosin IIA (COS-7 cells) stabilizes microtubules and slows cell migration. We also found that the microtubule kinesin Eg5 is essential for these regulatory effects of myosin IIA. These results establish a novel role for myosin IIA as a molecular down-regulator of the speed of cell migration, as well as identifying it as a novel crosstalk-regulator of tubulin, the Rac signaling system, and cell migration. [unreadable] [unreadable] A particularly intensively studied mediator of integrin cytoskeletal and signaling interactions is the scaffold protein termed focal adhesion kinase (FAK). We had previously published a series of studies on FAK concerning its potential roles in adhesion and migration in normal and tumor cells. We are collaborating with Hilary Beggs at UCSF and Joseph Schlessinger at Yale to determine roles of FAK and its homologue Pyk2 in cell migration in two- and three-dimensional microenvironments and other processes using conditional knockout cells. [unreadable] [unreadable] We had initiated a relatively high-risk project 5 years ago to develop new imaging methods to visualize actin and integrin dynamics at the extreme leading edge of migrating cells, particularly in lamellipodia and filopodia -- the thin extensions of the lamella of migrating cells. The goal was to determine how migrating cells such as fibroblasts and neurons produce local cell protrusions involved in cell migration and how they probe their local microenvironment to establish the very first attachments to the substrate. This goal was achieved using a combination of high-resolution confocal visualization with quantification of actin and integrin dynamics using optical and quantitative image analyses including local photoactivation of actin fluorescence. Working with the Carl Zeiss microscope company and in collaboration with James Galbraith at NINDS, new imaging and quantification algorithms have been developed. Our mechanistic cell biological studies reveal that local foci of actin polymerization shuttle rapidly transversely along the leading edge of migrating cells to make "sticky fingers," i.e. local membrane protrusions with integrins clustered into tiny precursors of adhesions that are locally activated to bind fibronectin. These tiny cellular sticky fingers probe back and forth to search for favorable sites for potential formation of new cell adhesions by fibroblasts and neurons. These findings establish the concept that actin dynamics regulate the sites of forward cell advance and subsequent potential attachment via integrins during pathfinding by migrating cells.[unreadable] [unreadable] These ongoing studies on the functions of integrins and associated intracellular and extracellular molecules in cell migration center upon our ability to image live-cell molecular dynamics of early cell protrusions and intracellular myosin and microtubules. All of these processes will need to be analyzed in parallel in real time to be able to understand the mechanisms of in vivo cell migration. This combined knowledge should provide novel approaches to understanding, preventing, or ameliorating migratory processes that cells use in abnormal development and cancer. An in-depth understanding of exactly how cells move and interact with their matrix environment will also facilitate tissue engineering studies.[unreadable] [unreadable] In summary, our Section is elucidating mechanisms by which integrins and cytoskeletal molecules function in cell adhesion and migration. A unifying theme has been the importance of understanding the dynamics of local cell-matrix-cytoskeletal interactions. Such analyses are important for understanding how adhesion receptors control cell and tissue movements, organization, growth, and development. We will continue to search for novel mechanisms and modulators. Knowledge of these basic processes should facilitate creative approaches to therapy, particularly in the fields of tissue engineering and cancer biology.